Abstract
Purpose :
Human RPE cells contain numerous lipofuscin (LF), melanolipofuscin (ML), and melanosome (M) organelles that vary in number with retinal location (fovea, perifovea, and near periphery) and age (PMID: 32433758) as well as impact on clinical autofluorescence (AF) imaging. Here, we examined the effect of AMD on granule count and AF of RPE cell bodies.
Methods :
Seven AMD-affected human RPE flatmounts (early: 3, late dry: 1; neovascular: 3) were imaged at three locations (fovea, perifovea, near periphery) using structured illumination microscopy (SIM) and confocal AF (both: excitation 488 nm). Z-stacks (step size: SIM 100 nm, confocal 390nm) were acquired from apical to basal through RPE cell bodies of 10 adjacent cells in each region. Subsequently, LF, ML, and M were marked, counted, and classified based on their AF properties using a customized ImageJ plug-in. AF/cell was calculated from confocal images. Furthermore, we analysed the impact of AMD on the total number of granules and AF/cell implementing a mixed effect ANCOVA, correlated granule count with cell size, and compared the results with data from normal RPE cells (PMID: 32433758).
Results :
Of 152 RPE cells analyzed, average granule count/cell was 449 ± 276 LF, 532 ± 377 ML, and 4 ± 2 M, with early AMD demonstrating higher average granule count (987 ± 616) and lower LF (406 ± 287) compared with late-stage AMD manifestations. While there was a high variation in granule load among cells, the foveal RPE cells had lowest LF (194 ± 290) and total granule count (851 ± 917). AMD-affected RPE cells were larger than RPE cells in normal eyes, had a higher granule density (granules/µm2), but showed significantly lower AF at all three locations (fovea: < 0.01; perifovea: 0.02; near periphery: <0.01).
Conclusions :
The reduced histological AF signal in AMD eyes compared to healthy cells mirrors and helps to explain clinical AMD AF imaging. Enlarged AMD affected RPE cells might reflect cell fusion or failed cytokinesis (PMID 26875723), which increases net granule count and diminishes total AF. Interestingly, LF was lowest at the fovea typically affected by accumulation of drusen in AMD as well as preservation of cone-mediated visual acuity. Here, we examined RPE cell bodies, but future studies should also focus on M within RPE’s apical processes and their impact on RPE AF imaging (PMID 32648890).
This is a 2021 ARVO Annual Meeting abstract.